In vitro, liver sinusoidal endothelial cells (LSECs) from healthy liver prevent hepatic stellate cell (HSC) activation and promote inactivation of activated HSC, but LSECs that have capillarized do not. Capillarization, the loss of LSEC fenestration and formation of a more organized basement membrane in the space of Disse, precedes fibrosis. In vivo, pharmacological reversal of capillarization after discontinuing a fibrotic insult accelerates inactivation of HSC and regression of fibrosis, whereas reversal of capillarization while a fibrotic stimulus is continued prevents progression of cirrhosis. Thus capillarization doesn't just precede fibrosis, but is permissive for hepatic fibrosis. The goals of this proposal are two-fold. First, examine how LSECs promote HSC quiescence. Second, elucidate the mechanisms that lead to capillarization. This proposal has three specific aims. In specific aim 1 the protein secreted by LSECs that promotes HSC quiescence will be identified, its in vivo activity will be confirmed, expression patterns within the liver will be immunolocalize, and its signaling within HSC will be examined. Specific 2 will examine the genesis of capillarized LSECs in a model of toxin-induced fibrosis and confirm that capillarized LSECs in a model of diet-induced non-alcoholic steatohepatitis have the same origin. Integrin expression, endocytosis and endocytosis receptors, and transcriptomic profiling in in vivo capillarized LSECs will be compared with LSECs from normal liver and in vitro capillarized LSECs to better understand capillarization. Preliminary data for specific aim 3 has identified a change within the fibrotic liver associated with loss of LSEC fenestration and with angiogenesis. Specific aim 3 will determine which type of liver cell is responsible for the change; confirm the association with loss of fenestration, HSC activation, fibrosis and angiogenesis; examine signaling pathways in the LSEC; and characterize the regulation of the change in the capillarized liver. Successful completion of these aims will transform our understanding of the mechanisms underlying capillarization and provide potential therapeutic targets to treat fibrosis.